Biochemical analysis unit

ABSTRACT

A biochemical analysis unit comprises a base plate, which has holes, and a porous adsorptive material, which is filled in each of the holes and forms each of adsorptive regions. Each of the adsorptive regions is provided with a layer, which has pores having a comparatively small mean pore diameter, and a layer, which has pores having a comparatively large mean pore diameter. In cases where the layers, which constitute each of the adsorptive regions, are connected with the layers, which constitute an adjacent adsorptive region, at one of surfaces of the base plate, the biochemical analysis unit further comprises a signal absorbing layer for absorbing a signal, which passes through layers located under the base plate and thus propagates from a certain hole toward an adjacent hole.

This is a divisional of application Ser. No. 10/800,676 filed Mar. 16,2004. The entire disclosures of the prior application, applicationnumber is Ser. No. 10/800,676 considered part of the disclosure of theaccompanying divisional application and is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a biochemical analysis unit for use in anoperation for detecting a receptor or a ligand by the utilization of alabeling substance.

2. Description of the Related Art

Heretofore, in the fields of clinical examinations, and the like,analyses of various samples have been made. Also, in order for quick andaccurate analyses of the samples to be made, various analysis implementsfor use in analysis kits and analysis instruments have been proposed.For example, in Japanese Patent No. 3298836, a sample analysis implementcomprising an analyzing section and a porous sample introducing section,which has a mean pore diameter larger than the mean pore diameter of theanalyzing section, is proposed. The proposed sample analysis implement,wherein the mean pore diameter of the porous sample introducing sectionis set to be large, and wherein the mean pore diameter of the analyzingsection is set to be small, enables the separation of constituents of asample, such that the sample may be processed quickly.

Also, in the fields of molecular biology, such as genetic expressionanalysis, macro arrays comprising a membrane and a plurality of spots(dots), which contain biopolymers, such as DNA's, and are arrayed on themembrane, have heretofore been known. With the macro arrays, multiplekinds of samples are capable of being analyzed at one time on a singlemembrane. Therefore, the macro arrays have heretofore been used widelyin the fields of molecular biology and the medical fields. For example,various kinds of DNA fragments (probes) may be arrayed in the form ofdots on the macro array, and a target, which has been prepared frommRNA, or the like, may be added onto the macro array. In this manner,hybridization, or the like, may be caused to occur. In such cases,behavior of a plurality of genes can be analyzed at one time.

The conventional macro arrays are constituted of a polymeric organicmembrane formed from nitrocellulose, or the like. Therefore, theconventional macro arrays are markedly soft and are apt to suffer frombending and creasing, which adversely affects the analytic operations,and the like. Accordingly, a macro array comprising a film-shaped hardporous body and a plurality of spots, which contain test substances andare arrayed on the film-shaped hard porous body, has been proposed in,for example, U.S. Pat. No. 6,492,119.

Also, various micro array analysis systems and various macro arrayanalysis systems have heretofore been used. With the micro arrayanalysis systems and the macro array analysis systems, liquidscontaining ligands or receptors (i.e., the substances, which are capableof specifically binding to organism-originating substances and whosebase sequences, base lengths, compositions, characteristics, and thelike, are known) are spotted onto different positions on a surface of abiochemical analysis unit, such as a membrane filter, and a plurality ofadsorptive regions are thereby formed on the surface of the biochemicalanalysis unit. Examples of the ligands or the receptors includehormones, tumor markers, enzymes, antibodies, antigens, abzymes, otherproteins, nucleic acids, cDNA's, DNA's, and RNA's. Thereafter, a labeledreceptor or a labeled ligand, which has been labeled with a radioactivelabeling substance, a fluorescent labeling substance, a labelingsubstance capable of causing a chemical luminescence substrate toproduce chemical luminescence when being brought into contact with thechemical luminescence substrate, or the like, is subjected tohybridization, or the like, with the ligands or the receptors, which arecontained in the adsorptive regions of the biochemical analysis unit.The labeled receptor or the labeled ligand is thus specifically bound toat least one of the ligands or the receptors, which are contained in theadsorptive regions of the biochemical analysis unit. The labeledreceptor or the labeled ligand is the substance, which has been sampledfrom an organism through extraction, isolation, or the like, or has beensubjected to chemical treatment after being sampled, and which has beenlabeled with the radioactive labeling substance, the fluorescentlabeling substance, the labeling substance capable of causing a chemicalluminescence substrate to produce the chemical luminescence when beingbrought into contact with the chemical luminescence substrate, or thelike. Examples of the labeled receptors or the labeled ligands includehormones, tumor markers, enzymes, antibodies, antigens, abzymes, otherproteins, nucleic acids, DNA's, and mRNA's.

In cases where the labeled receptor or the labeled ligand has beenlabeled with the radioactive labeling substance, a stimulable phosphorlayer of a stimulable phosphor sheet is then exposed to radiationradiated out from the radioactive labeling substance, which is containedselectively in the adsorptive regions of the biochemical analysis unit.Thereafter, the stimulable phosphor layer is exposed to stimulatingrays, which cause the stimulable phosphor layer to emit light inproportion to the amount of energy stored on the stimulable phosphorlayer during the exposure of the stimulable phosphor layer to theradiation. The light emitted by the stimulable phosphor layer isdetected photoelectrically, and data for a biochemical analysis isthereby obtained.

In cases where the labeled receptor or the labeled ligand has beenlabeled with the fluorescent labeling substance, excitation light isirradiated to the adsorptive regions of the biochemical analysis unit,and the fluorescent labeling substance, which is contained selectivelyin the adsorptive regions of the biochemical analysis unit, is excitedby the excitation light to produce fluorescence. The thus producedfluorescence is detected photoelectrically, and data for a biochemicalanalysis is thereby obtained.

In cases where the labeled receptor or the labeled ligand has beenlabeled with the labeling substance capable of causing a chemicalluminescence substrate to produce the chemical luminescence when beingbrought into contact with the chemical luminescence substrate, thelabeling substance, which is contained selectively in the adsorptiveregions of the biochemical analysis unit, is brought into contact withthe chemical luminescence substrate. Also, the chemical luminescenceproduced by the labeling substance is detected photoelectrically, anddata for a biochemical analysis is thereby obtained.

The micro array analysis systems and the macro array analysis systemsare described in, for example, U.S. Patent Laid-Open No. 20020061534.

With the micro array analysis systems and the macro array analysissystems described above, a large number of the adsorptive regions, towhich the ligands or the receptors are bound, are capable of beingformed at a high density at different positions on the surface of thebiochemical analysis unit, and the labeled receptor or the labeledligand, which has been labeled with the labeling substance, is capableof being subjected to the hybridization, or the like, with the ligandsor the receptors, which have been bound to the adsorptive regions formedat a high density at different positions on the surface of thebiochemical analysis unit. Therefore, the micro array analysis systemsand the macro array analysis systems described above have the advantagesin that a receptor or a ligand is capable of being analyzed quickly.

[Patent Literature 1]

Japanese Patent No. 3298836

[Patent Literature 2]

U.S. Pat. No. 6,492,119

[Patent Literature 3]

U.S. Patent Laid-Open No. 20020061534

However, the ligand or the receptor, which is bound to each of theadsorptive regions of the biochemical analysis unit described above, isbound and fixed to the entire area of each of the adsorptive regions.Therefore, the problems occur in that a signal coming from the receptoror the ligand having been bound to the ligand or the receptor havingbeen fixed to an area of each of the adsorptive regions, which area isremote from a detection surface, is attenuated. As for the micro arrayanalysis systems and the macro array analysis systems described above,it is desired that the receptor or the ligand be capable of beinganalyzed more accurately. However, the attenuation of the signaldescribed above obstructs the formation of accurate data for abiochemical analysis.

SUMMARY OF THE INVENTION

The primary object of the present invention is to provide a biochemicalanalysis unit, which enables attenuation of a signal coming from areceptor or a ligand to be suppressed.

In Patent Literature 2 described above, it is described that thefilm-shaped hard porous body is constituted of a surface layer region,which is provided with through-pores having a comparatively small meanpore diameter, and a base layer region, which is provided withthrough-pores having a comparatively large mean pore diameter. However,the film-shaped hard porous body described in Patent Literature 2 shouldbe distinctly distinguished from a biochemical analysis unit inaccordance with the present invention comprising a base plate, which hasa plurality of holes, and a porous adsorptive material, which is filledin each of the plurality of the holes of the base plate. Further, theconstitution described in Patent Literature 2, wherein the film-shapedhard porous body is constituted of the surface layer region, which isprovided with the through-pores having a comparatively small mean porediameter, and the base layer region, which is provided with thethrough-pores having a comparatively large mean pore diameter, is theconstitution which aims at the effect such that a liquid containing aprobe may quickly penetrate into the film-shaped hard porous body whenthe liquid containing the probe is spotted onto the film-shaped hardporous body. The constitution of the film-shaped hard porous body aimingat the effect described above is clearly different from the constitutionof the biochemical analysis unit in accordance with the presentinvention.

The present invention provides a first biochemical analysis unit,comprising:

i) a base plate, which has a plurality of holes, and

ii) a porous adsorptive material, which is filled in each of theplurality of the holes of the base plate and forms each of a pluralityof adsorptive regions,

wherein each of the adsorptive regions is provided with a layer, whichhas pores having a comparatively small mean pore diameter, and a layer,which has pores having a comparatively large mean pore diameter.

The term “pore diameter” as used herein means the mean value of thevalues of the longest diameter and the shortest diameter of a certainhole. The difference in mean pore diameter between the layer, which hasthe pores having a comparatively small mean pore diameter, and thelayer, which has the pores having a comparatively large mean porediameter, may vary in accordance with the layer thickness of the layer,which has the pores having a comparatively small mean pore diameter, thelayer thickness of the layer, which has the pores having a comparativelylarge mean pore diameter, or the like. However, in cases where the meanpore diameter of the pores of the layer, which has the pores having acomparatively large mean pore diameter, is taken as 1, the mean porediameter of the pores of the layer, which has the pores having acomparatively small mean pore diameter, should preferably be at most0.7, should more preferably be at most 0.5, and should most preferablybe at most 0.4.

The first biochemical analysis unit in accordance with the presentinvention should preferably be modified such that the layers, whichconstitute each of the adsorptive regions, are connected with thelayers, which constitute an adjacent adsorptive region, at one ofsurfaces of the base plate, and

the biochemical analysis unit further comprises a signal absorbing layerfor absorbing a signal, which passes through layers located under thebase plate and thus propagates from a certain hole of the base platetoward an adjacent hole of the base plate.

The present invention also provides a second biochemical analysis unit,comprising:

i) abase plate, which has a plurality of holes, and

ii) a porous adsorptive material, which is filled in each of theplurality of the holes of the base plate and forms each of a pluralityof adsorptive regions,

wherein each of the adsorptive regions is provided with a layerconstituted of a material having a comparatively large quantity of afunctional group, which is capable of binding with a ligand or areceptor to be bound to the adsorptive region, and a layer constitutedof a material having a comparatively small quantity of a functionalgroup, which is capable of binding with the ligand or the receptor to bebound to the adsorptive region.

The kind of the functional group, which is capable of binding with theligand or the receptor to be bound to the adsorptive region, may vary inaccordance with the kind of the ligand or the receptor, which is to bebound to the adsorptive region. Examples of the functional groupsinclude a carboxyl group, an amino group, an amido group which becomescapable of binding with the ligand or the receptor when being exposed toultraviolet light, a group for forming an ester linkage which becomescapable of binding with the ligand or the receptor when being subjectedto saponification treatment, and a hydroxyl group which becomes capableof binding with the ligand or the receptor by the aid of a silanecoupling agent.

The difference between the quantity of the functional group, which iscontained in the layer constituted of the material having acomparatively large quantity of the functional group, and the quantityof the functional group, which is contained in the layer constituted ofthe material having a comparatively small quantity of the functionalgroup, may vary in accordance with the layer thickness of the layerconstituted of the material having a comparatively large quantity of thefunctional group, the layer thickness of the layer constituted of thematerial having a comparatively small quantity of the functional group,the kind of the functional group, or the like. However, in cases wherethe density of the functional group in the layer constituted of thematerial having a comparatively large quantity of the functional groupis taken as 1, the density of the functional group in the layerconstituted of the material having a comparatively small quantity of thefunctional group should preferably be at most 0.7, should morepreferably be at most 0.5, and should most preferably be at most 0.4.

The second biochemical analysis unit in accordance with the presentinvention should preferably be modified such that the layers, whichconstitute each of the adsorptive regions, are connected with thelayers, which constitute an adjacent adsorptive region, at one ofsurfaces of the base plate, and

the biochemical analysis unit further comprises a signal absorbing layerfor absorbing a signal, which passes through layers located under thebase plate and thus propagates from a certain hole of the base platetoward an adjacent hole of the base plate.

The first biochemical analysis unit in accordance with the presentinvention comprises the base plate, which has the plurality of theholes, and the porous adsorptive material, which is filled in each ofthe plurality of the holes of the base plate and forms each of theplurality of the adsorptive regions. Also, each of the adsorptiveregions is provided with the layer, which has the pores having acomparatively small mean pore diameter, and the layer, which has thepores having a comparatively large mean pore diameter. Therefore, withthe first biochemical analysis unit in accordance with the presentinvention, the layer, which has the pores having a comparatively smallmean pore diameter, has a large specific surface area and is capable ofefficiently immobilizing the ligand or the receptor. Also, the layer,which has the pores having a comparatively large mean pore diameter, iscapable of uniformly distributing a reaction liquid over the entire areaof the biochemical analysis unit and enhancing the self-supportingcharacteristics of the porous adsorptive material. Specifically, a partof the functions of the adsorptive region is capable of being assignedto the layer, which has the pores having a comparatively small mean porediameter, and the other part of the functions of the adsorptive regionis capable of being assigned to the layer, which has the pores having acomparatively large mean pore diameter.

The ligand or the receptor, which is to be immobilized, converges uponthe layer, which has the pores having a comparatively small mean porediameter. Therefore, in cases where a receptor or a ligand, which hasbeen specifically bound to the immobilized ligand or the immobilizedreceptor, is to be detected by the utilization of a signal coming from alabeling substance, the detection of the receptor or the ligand may beperformed from the side of the layer, which has the pores having acomparatively small mean pore diameter. In such cases, the detection ofthe receptor or the ligand is capable of being performed such that thesignal coming from the receptor or the ligand may not be attenuated.

The second biochemical analysis unit in accordance with the presentinvention comprises the base plate, which has the plurality of theholes, and the porous adsorptive material, which is filled in each ofthe plurality of the holes of the base plate and forms each of theplurality of the adsorptive regions. Also, each of the adsorptiveregions is provided with the layer constituted of the material having acomparatively large quantity of the functional group, which is capableof binding with a ligand or a receptor to be bound to the adsorptiveregion, and the layer constituted of the material having a comparativelysmall quantity of the functional group, which is capable of binding withthe ligand or the receptor to be bound to the adsorptive region.Therefore, with the second biochemical analysis unit in accordance withthe present invention, the layer constituted of the material having acomparatively large quantity of the functional group is capable ofefficiently immobilizing the ligand or the receptor. Also, the layerconstituted of the material having a comparatively small quantity of thefunctional group is capable of uniformly distributing the reactionliquid over the entire area of the biochemical analysis unit andenhancing the self-supporting characteristics of the porous adsorptivematerial. Further, the layer constituted of the material having acomparatively small quantity of the functional group is capable ofpreventing the problems from occurring in that the receptor or theligand undergoes non-specific adsorption by an electrostatic interactionor a polar interaction. Specifically, a part of the functions of theadsorptive region is capable of being assigned to the layer constitutedof the material having a comparatively large quantity of the functionalgroup, and the other part of the functions of the adsorptive region iscapable of being assigned to the layer constituted of the materialhaving a comparatively small quantity of the functional group.

The ligand or the receptor, which is to be immobilized, converges uponthe layer constituted of the material having a comparatively largequantity of the functional group. Therefore, in cases where the receptoror the ligand, which has been specifically bound to the immobilizedligand or the immobilized receptor, is to be detected by the utilizationof the signal coming from the labeling substance, the detection of thereceptor or the ligand may be performed from the side of the layerconstituted of the material having a comparatively large quantity of thefunctional group. In such cases, the detection of the receptor or theligand is capable of being performed such that the signal coming fromthe receptor or the ligand may not be attenuated.

Each of the first biochemical analysis unit and the second biochemicalanalysis unit in accordance with the present invention may be modifiedsuch that the layers, which constitute each of the adsorptive regions,are connected with the layers, which constitute an adjacent adsorptiveregion, at one of surfaces of the base plate, and such that thebiochemical analysis unit further comprises the signal absorbing layerfor absorbing the signal, which passes through the layer located underthe base plate and thus propagates from a certain hole of the base platetoward an adjacent hole of the base plate. With each of the modificationof the first biochemical analysis unit in accordance with the presentinvention and the modification of the second biochemical analysis unitin accordance with the present invention, the problems are capable ofbeing efficiently prevented from occurring in that the signal comingfrom the receptor or the ligand, which is to be detected from a certainhole of the base plate, passes through the layers, which constitute eachof the adsorptive regions and are connected with the layers constitutingan adjacent adsorptive region at the position under the base plate, andthe signal thus propagates toward an adjacent hole of the base plate.Accordingly, only the signal, which comes from the receptor or theligand that is to be detected, is capable of being detected, and thesignal coming from each of the holes of the base plate is capable ofbeing detected accurately.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic perspective view showing an embodiment of thebiochemical analysis unit in accordance with the present invention,

FIG. 2 is a schematic sectional view showing a part of the embodiment ofthe biochemical analysis unit in accordance with the present invention,

FIGS. 3A and 3B are schematic views showing an example of how thebiochemical analysis unit in accordance with the present invention isproduced,

FIG. 4 is a schematic view showing a different example of how thebiochemical analysis unit in accordance with the present invention isproduced, and

FIG. 5 is a schematic sectional view showing an example of a reactor, inwhich a reaction liquid is forcibly caused to flow.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will hereinbelow be described in further detailwith reference to the accompanying drawings.

FIG. 1 is a schematic perspective view showing an embodiment of thebiochemical analysis unit in accordance with the present invention. Withreference to FIG. 1, a biochemical analysis unit 1 comprises a baseplate 2, which is provided with a plurality of holes 3, 3, . . . , and aplurality of adsorptive regions 4, 4, . . . , each of which is filled inone of the holes 3, 3, . . . and comprises a porous material adhered tothe base plate 2.

FIG. 2 is a schematic sectional view showing a part of the embodiment ofthe biochemical analysis unit in accordance with the present invention.As illustrated in FIG. 2, each of the adsorptive regions 4, 4, . . . isconstituted of a layer 4 a, which has pores having a comparatively smallmean pore diameter, and a layer 4 b, which has pores having acomparatively large mean pore diameter. Also, a signal absorbing layer 5is formed under the layer 4 b, which has the pores having acomparatively large mean pore diameter. The signal absorbing layer 5absorbs a signal, which passes through an underside area 6 located underthe base plate 2 and thus propagates from a hole 3 a of the base plate 2toward an adjacent hole 3 b of the base plate 2. At the underside area 6located under the base plate 2, the layer 4 a and the layer 4 b, whichconstitute each of the adsorptive regions 4, 4, . . . , are connectedwith the layer 4 a and the layer 4 b, which constitute an adjacentadsorptive region 4. Specifically, the underside area 6 located underthe base plate 2 is constituted of the layer 4 a, the layer 4 b, and thesignal absorbing layer 5, which have been compressed by being pressedtogether against the bottom surface of the base plate 2.

A ligand or a receptor, which is to be fixed to each of the adsorptiveregions 4, 4, . . . , converges upon the layer 4 a, which has the poreshaving a comparatively small mean pore diameter, and is scarcely boundto the layer 4 b, which has the pores having a comparatively large meanpore diameter. With the biochemical analysis unit 1, the ligand or thereceptor, which is fixed to each of the adsorptive regions 4, 4, . . . ,converges upon the layer 4 a, which has the pores having a comparativelysmall mean pore diameter. Therefore, in cases where a receptor or aligand, which has been specifically bound to the ligand or the receptorhaving been fixed to each of the adsorptive regions 4, 4, . . . , is tobe detected by the utilization of a labeling substance, the detection ofthe receptor or the ligand may be performed from the side of the layer 4a, which has the pores having a comparatively small mean pore diameter.In such cases, the detection of the receptor or the ligand is capable ofbeing performed such that the signal coming from the receptor or theligand may not be attenuated.

Also, with the biochemical analysis unit 1, at the underside area 6located under the base plate 2, the layer 4 a and the layer 4 b, whichconstitute each of the adsorptive regions 4, 4, . . . , are connectedwith the layer 4 a and the layer 4 b, which constitute an adjacentadsorptive region 4. Therefore, there is possibility of the signalpropagating from the hole 3 a toward the adjacent hole 3 b. However,with the biochemical analysis unit 1, wherein the ligand or thereceptor, which is to be fixed to each of the adsorptive regions 4, 4, .. . , converges upon the layer 4 a, which has the pores having acomparatively small mean pore diameter, the amount of the signalpropagating from the hole 3 a toward the adjacent hole 3 b is capable ofbeing kept smaller than with a biochemical analysis unit, wherein theligand or the receptor is bound in a dispersed form over the entire areaof the adsorptive region. Further, since the biochemical analysis unit 1is provided with the signal absorbing layer 5, the signal propagatingfrom the hole 3 a toward the adjacent hole 3 b is capable of beingefficiently absorbed by the signal absorbing layer 5. Accordingly, theeffect of the signal propagating from the hole 3 a toward the adjacenthole 3 b is capable of being suppressed.

In FIG. 2, each of the adsorptive regions 4, 4, . . . is constituted ofthe two layers, i.e. the layer 4 a, which has the pores having acomparatively small mean pore diameter, and the layer 4 b, which has thepores having a comparatively large mean pore diameter. However, thenumber of the layers constituting each of the adsorptive regions 4, 4, .. . is not limited to two. For example, the biochemical analysis unit 1in accordance with the present invention may be modified such that eachof the adsorptive regions 4, 4, . . . is constituted of the layer 4 a,which has the pores having a comparatively small mean pore diameter, thelayer 4 b, which has the pores having a comparatively large mean porediameter, and an intermediate layer, which is formed between the layer 4a and the layer 4 b and has pores having a mean pore diameter fallingbetween the comparatively small mean pore diameter of the pores of thelayer 4 a and the comparatively large mean pore diameter of the pores ofthe layer 4 b.

Also, in FIG. 2, the signal absorbing layer 5 is formed over the entirearea of the bottom surface of the base plate 2. Alternatively, thesignal absorbing layer may be formed between the layer 4 a and the layer4 b. As another alternative, the signal absorbing layer 5 may be formedonly at the position exactly under the layer 4 b so as to close each ofthe holes 3, 3, . . . As a further alternative, the signal absorbinglayer 5 may be formed only at the position of the underside area 6, atwhich the layer 4 a, the layer 4 b, and the signal absorbing layer 5have been compressed together, such that the signal absorbing layer 5 iscapable of absorbing the signal propagating from a certain hole to anadjacent hole. As a still further alternative, the signal absorbinglayer 5 may be formed only at a certain part of the underside area 6, atwhich the layer 4 a, the layer 4 b, and the signal absorbing layer 5have been compressed together, such that the signal absorbing layer 5 iscapable of absorbing the signal propagating from a certain hole to anadjacent hole.

Such that light scattering may be prevented from occurring within thebiochemical analysis unit 1, the base plate 2 should preferably be madefrom a material, which does not transmit radiation or light, or whichattenuates radiation or light. The material for the formation of thebase plate 2 should preferably be a metal or a ceramic material. Also,in cases where a plastic material, for which the hole making processingis capable of being performed easily, is employed as the material forthe formation of the base plate 2, particles should preferably bedispersed within the plastic material, such that radiation or light iscapable of being attenuated even further.

Examples of the metals, which may be utilized preferably for theformation of the base plate 2, include copper, silver, gold, zinc, lead,aluminum, titanium, tin, chromium, iron, nickel, cobalt, tantalum, andalloys, such as stainless steel and bronze. Examples of the ceramicmaterials, which may be utilized preferably for the formation of thebase plate 2, include alumina, zirconia, magnesia, and quartz. Examplesof the plastic materials, which may be utilized preferably for theformation of the base plate 2, include polyolefins, such as apolyethylene and a polypropylene; polystyrenes; acrylic resins, such asa polymethyl methacrylate; polyvinyl chlorides; polyvinylidenechlorides; polyvinylidene fluorides; polytetrafluoroethylenes;polychlorotrifluoroethylenes; polycarbonates; polyesters, such as apolyethylene naphthalate and a polyethylene terephthalate; aliphaticpolyamides, such as a 6-nylon and a 6,6-nylon; polyimides; polysulfones;polyphenylene sulfides; silicon resins, such as a polydiphenyl siloxane;phenolic resins, such as novolak; epoxy resins; polyurethanes;celluloses, such as cellulose acetate and nitrocellulose; copolymers,such as a butadiene-styrene copolymer; and blends of plastic materials.

Also, in cases where the receptor or the ligand, which is to bedetected, is subjected to the specific binding with the ligands or thereceptors, each of which has been bound to one of the adsorptive regions4, 4, . . . , and the receptor or the ligand, which has thus been boundto at least one of the ligands or the receptors having been bound to theadsorptive regions 4, 4, . . . , is to be detected by the utilization ofthe labeling substance, it is desired that the radiation or the lightradiated out from the labeling substance within a hole 3 of the baseplate 2 be prevented from passing from the hole 3 through the base platewall to the adjacent hole 3. Therefore, in cases where the base plate 2is constituted of a plastic material, in order for the radiation or thelight to be attenuated, the plastic material should preferably be loadedwith particles of metal oxides, glass fibers, or the like. Examples ofthe metal oxides include silicon dioxide, alumina, titanium dioxide,iron oxide, and copper oxide. However, the metal oxides are not limitedto those enumerated above.

The radiation attenuating properties or the light attenuating propertiesshould preferably be such that, when the radiation or the light, whichis radiated out from the labeling substance within the hole 3, haspassed from the hole 3 through the base plate wall to the adjacent hole3, the intensity of the radiation or the light reduces to an intensityof at most ⅕ of the original intensity. The radiation attenuatingproperties or the light attenuating properties should more preferably besuch that the intensity of the radiation or the light having passedthrough the base plate wall in the manner described above reduces to anintensity of at most 1/10 of the original intensity.

In order for the radiation, such as electron rays, coming from aradioactive labeling substance to be blocked efficiently, the meandensity of the base plate 2 may ordinarily be at least 0.6 g/cm³. Themean density of the base plate 2 should preferably fall within the rangeof 1 g/cm³ to 20 g/cm³, and should more preferably fall within the rangeof 2 g/cm³ to 10 g/cm³. Since the transmission distance of the electronrays is in inverse proportion to the density, in cases where theradioactive labeling substance is an ordinary radioactive isotope (RI),such as ³²P, ³³P, ³⁵S, or ¹⁴C, and the mean density of the base plate 2falls within the range described above, the electron rays coming fromthe RI of the sample, which is fixed within each of the holes 3, 3, . .. , is capable of being blocked by the partition wall of the base plate2, and the problems are capable of being prevented from occurring inthat resolution of a radiation image is adversely affected bytransmission and scattering of the electron rays.

The thickness of the base plate 2 may ordinarily fall within the rangeof 50 μm to 1,000 μm, and should preferably fall within the range of 100μm to 500 μm.

Such that the density of the holes 3, 3, . . . made through the baseplate 2 may be enhanced, the area (size) of the opening of each of theholes 3, 3, . . . may ordinarily be smaller than 5 mm². The area of theopening of each of the holes 3, 3, . . . should preferably be smallerthan 1 mm², should more preferably be smaller than 0.3 mm², and shouldmost preferably be smaller than 0.01 mm². Also, the area of the openingof each of the holes 3, 3, . . . should preferably be at least 0.001mm².

The pitch of the holes 3, 3, . . . (i.e., the distance between thecenter points of two holes which are adjacent to each other) shouldpreferably fall within the range of 0.05 mm to 3 mm. Also, the spacingbetween two adjacent holes 3, 3 (i.e., the shortest distance betweenedges of two adjacent holes 3, 3) should preferably fall within therange of 0.01 mm to 1.5 mm. The number (the array density) of the holes3, 3, . . . may ordinarily be at least 10 holes/cm². The number (thearray density) of the holes 3, 3, . . . should preferably be at least100 holes/cm², should more preferably be at least 500 holes/cm², andshould most preferably be at least 1,000 holes/cm². Also, the number(the array density) of the holes 3, 3, . . . should preferably be atmost 100,000 holes/cm², and should more preferably be at most 10,000holes/cm². The holes 3, 3, . . . need not necessarily be arrayed atequal spacing as illustrated in FIG. 1. For example, the holes 3, 3, . .. may be grouped into several number of blocks (units) comprising aplurality of holes and may be formed in units of the blocks.

Perforation of the plurality of the holes 3, 3, . . . through the baseplate 2 may be performed with, for example, a punching technique forpunching with a pin, a technique for electrical discharge machining, inwhich a pulsed high voltage is applied across electrodes in order tovolatilize the base plate material, an etching technique, or a laserbeam irradiation technique. In cases where the material of the baseplate is a metal material or a plastic material, the biochemicalanalysis unit may be prepared with an operation for performing coronadischarge or plasma discharge on the surface of the base plate, applyingan adhesive agent to the surface of the base plate, and laminating theporous material for the formation of the adsorptive regions by use ofmeans, such as a press. The adhesive agent described above shouldpreferably be styrene-butadiene rubber, acrylonitrile-butadiene rubber,or the like. The application of the adhesive agent should preferably beperformed with a roll coating technique, a wire bar coating technique, adip coating technique, a blade coating technique, or the like. Also, incases where the porous material for the formation of the adsorptiveregions is pressed against the base plate, the base plate and the porousmaterial for the formation of the adsorptive regions may be dividedpreviously into a plurality of sheets, and the plurality of the sheetsmay be pressed intermittently. Alternatively, a long web of the baseplate and a long web of the porous material for the formation of theadsorptive regions may be conveyed continuously between two rolls.

In the biochemical analysis unit in accordance with the presentinvention, as the porous material for the formation of the adsorptiveregions of the biochemical analysis unit, a porous quality material or afiber material may be utilized preferably. The porous quality materialand the fiber material may be utilized in combination in order to formthe adsorptive regions of the biochemical analysis unit. In thebiochemical analysis unit in accordance with the present invention, theporous material, which may be utilized for the formation of theadsorptive regions of the biochemical analysis unit, may be an organicmaterial, an inorganic material, or an organic-inorganic compositematerial.

The organic porous quality material, which may be utilized for theformation of the adsorptive regions of the biochemical analysis unit,may be selected from a wide variety of materials. However, the organicporous quality material should preferably be a carbon porous qualitymaterial, such as active carbon, or a porous quality material capable offorming a membrane filter. As the porous quality material capable offorming a membrane filter, a polymer soluble in a solvent shouldpreferably be utilized. Examples of the polymers soluble in a solventinclude cellulose derivatives, such as nitrocellulose, regeneratedcellulose, cellulose acetate, and cellulose acetate butyrate; aliphaticpolyamides, such as a 6-nylon, a 6,6-nylon, and a 4,10-nylon;polyolefins, such as a polyethylene and a polypropylene;chlorine-containing polymers, such as a polyvinyl chloride and apolyvinylidene chloride; fluorine resins, such as a polyvinylidenefluoride and a polytetrafluoride; polycarbonates; polysulfones; alginicacids and alginic acid derivatives, such as alginic acid, calciumalginate, and an alginic acid-polylysine polyion complex; and collagen.Copolymers or composite materials (mixture materials) of theabove-enumerated polymers may also be utilized.

The fiber material, which may be utilized for the formation of theadsorptive regions of the biochemical analysis unit, may be selectedfrom a wide variety of materials. Examples of the fiber materials, whichmay be utilized preferably, include the cellulose derivatives and thealiphatic polyamides enumerated above.

The inorganic porous quality material, which may be utilized for theformation of the adsorptive regions of the biochemical analysis unit,may be selected from a wide variety of materials. Examples of theinorganic porous quality materials, which may be utilized preferably,include metals, such as platinum, gold, iron, silver, nickel, andaluminum; oxides of metals, and the like, such as alumina, silica,titania, and zeolite; metal salts, such as hydroxyapatite and calciumsulfate; and composite materials of the above-enumerated materials.

After the porous adsorptive material for the formation of the adsorptiveregions of the biochemical analysis unit has been selectedappropriately, the layer, which has the pores having a comparativelysmall mean pore diameter, and the layer, which has the pores having acomparatively large mean pore diameter, are formed. Alternatively, alayer constituted of a material having a comparatively large quantity ofa functional group, which is capable of binding with a ligand or areceptor to be bound to the adsorptive region, and a layer constitutedof a material having a comparatively small quantity of a functionalgroup, which is capable of binding with the ligand or the receptor to bebound to the adsorptive region, may be formed.

Also, the signal absorbing layer is capable of being formed with aprocess, wherein a substance capable of absorbing the signal, such as alight signal or a radiation signal, is mixed with the porous adsorptivematerial described above. In cases where the signal is a chemicalluminescence signal or a fluorescence signal, the signal absorbing layeris capable of being formed with a process, wherein a dye, or the like,capable of absorbing light having wavelengths falling within thewavelength range of the chemical luminescence or the fluorescence ismixed with the porous adsorptive material described above. In caseswhere the signal is the radiation signal, the signal absorbing layer iscapable of being formed with a process, wherein fine particles of aheavy metal, such as lead or tungsten, which acts as a radiationblocking substance, are mixed with the porous adsorptive materialdescribed above.

In order for each of the adsorptive regions to be formed from the layer,which has the pores having a comparatively small mean pore diameter, andthe layer, which has the pores having a comparatively large mean porediameter, the layers may be formed with a process described below. Also,in order for each of the adsorptive regions to be formed from the layerconstituted of the material having a comparatively large quantity of thefunctional group, which is capable of binding with the ligand or thereceptor to be bound to the adsorptive region, and the layer constitutedof the material having a comparatively small quantity of the functionalgroup, which is capable of binding with the ligand or the receptor to bebound to the adsorptive region, the layers may be formed with a processdescribed below. Specifically, solutions (hereinbelow referred to as thedopes) containing different kinds of porous quality materials insolvents are successively cast or coated on a support, and the resultingcasting layers or the resulting coating layers are then dipped in badsolvents for the polymers of the porous films or in a mixed solvent ofgood solvents and bad solvents for the polymers and thereafter subjectedto washing with water and drying. Alternatively, the format ion of thelayers for forming each of the adsorptive regions may be formed with adifferent process, wherein one of different kinds of dopes is cast orcoated on a support, the other dope is cast or coated on a differentsupport, and each of the resulting casting layers or each of theresulting coating layers is then dried little by little.

FIGS. 3A and 3B are schematic views showing an example of how thebiochemical analysis unit in accordance with the present invention isproduced. In the example shown in FIGS. 3A and 3B, the biochemicalanalysis unit is produced with a pressing technique, wherein a porousfilm 21, which comprises two layers having pores having different meanpore diameters, and the base plate 2 are superposed one upon the otherand pressed together, and the porous film 21 is thereby press-fittedinto the holes 3, 3, . . . of the base plate 2. With the pressingtechnique, the porous film 21 is capable of being press-fitted into theholes 3, 3, . . . of the base plate 2 such that little change occurswith the pore diameters of the pores of the region of the porous film21, which region is press-fitted into each of the holes 3, 3, . . .

As illustrated in FIG. 3A, the porous film 21 and the base plate 2having the holes 3, 3, . . . are superposed one upon the other andpressed together by being passed between a press roll 22 and a back-uproll 23. In this manner, as illustrated in FIG. 3B, the porous film 21is press-fitted into the holes 3, 3, . . . of the base plate 2. In suchcases, the porous film 21 may be softened with a technique wherein, forexample, the press roll 22 and the back-up roll 23 are heated.

Alternatively, the biochemical analysis unit in accordance with thepresent invention may be produced with a technique, wherein the dopesare injected into the holes 3, 3, . . . of the base plate 2. FIG. 4 is aschematic view showing a different example of how the biochemicalanalysis unit in accordance with the present invention is produced. Asillustrated in FIG. 4, a dispenser 30 for injecting a dope 31 into theholes 3, 3, . . . of the base plate 2 and a dispenser 32 for injecting adope 33 into the holes 3, 3, . . . of the base plate 2 are located abovethe base plate 2, which is conveyed continuously or intermittently. InFIG. 4, as an aid in clarifying the relationship between each of thedopes 31 and 33 and the formation of the corresponding layer, thecontents of each of the dopes 31 and 33 are shown by the illustrationidentical with the illustration of the corresponding layer. Thedispenser 30 intermittently injects the dope 31 into each of the holes3, 3, . . . of the base plate 2. Thereafter, the dispenser 32intermittently injects the dope 33 onto the dope 31 having been injectedinto each of the holes 3, 3, . . . of the base plate 2. After the dopes31 and 33 have been injected into each of the holes 3, 3, . . . of thebase plate 2, air having a controlled temperature and a controlledhumidity is fed over the base plate 2 at a predetermined flow rate, andthe solvents contained in the dopes 31 and 33 are vaporized little bylittle. In this manner, the two different kinds of the layers arecapable of being formed.

The biochemical analysis unit in accordance with the present inventionis applicable broadly to various assay processes for:

i) obtaining a biochemical analysis unit provided with a plurality ofporous adsorptive regions, to which ligands or receptors have been boundrespectively,

ii) subjecting a reaction liquid containing at least one kind of areceptor or at least one kind of a ligand to specific binding with theligands or the receptors, each of which has been bound to one of theporous adsorptive regions of the biochemical analysis unit, the receptoror the ligand being thereby specifically bound to at least one of theligands, each of which has been bound to one of the porous adsorptiveregions of the biochemical analysis unit, or at least one of thereceptors, each of which has been bound to one of the porous adsorptiveregions of the biochemical analysis unit, and

iii) detecting the receptor or the ligand, which has thus beenspecifically bound to at least one of the ligands or at least one of thereceptors, by the utilization of a labeling substance.

In a first aspect, the biochemical analysis unit in accordance with thepresent invention is applicable to an assay process for:

i) obtaining a biochemical analysis unit provided with a plurality ofporous adsorptive regions, to which ligands or receptors have been boundrespectively,

ii) subjecting a reaction liquid containing at least one kind of alabeled receptor or at least one kind of a labeled ligand, which hasbeen labeled with a labeling substance, to specific binding with theligands or the receptors, each of which has been bound to one of theporous adsorptive regions of the biochemical analysis unit, the labeledreceptor or the labeled ligand being thereby specifically bound to atleast one of the ligands, each of which has been bound to one of theporous adsorptive regions of the biochemical analysis unit, or at leastone of the receptors, each of which has been bound to one of the porousadsorptive regions of the biochemical analysis unit, and

iii) detecting the labeled receptor or the labeled ligand, which hasthus been specifically bound to at least one of the ligands or at leastone of the receptors.

In such cases, the labeled receptor or the labeled ligand is thesubstance, which has been sampled from an organism through extraction,isolation, or the like, or has been subjected to chemical treatmentafter being sampled, and which has been labeled with the labelingsubstance. The labeled receptor or the labeled ligand is capable ofundergoing the specific binding with at least one of the ligands, eachof which has been bound to one of the porous adsorptive regions of thebiochemical analysis unit, or at least one of the receptors, each ofwhich has been bound to one of the porous adsorptive regions of thebiochemical analysis unit. Examples of the labeled receptors or thelabeled ligands include hormones, tumor markers, enzymes, antibodies,antigens, abzymes, other proteins, nucleic acids, DNA's, and mRNA's.

Examples of the labeling substances include a radioactive labelingsubstance, a fluorescent labeling substance, and a labeling substancecapable of causing a chemical luminescence substrate to produce thechemical luminescence when being brought into contact with the chemicalluminescence substrate. The labeling substance may be a substance, whichis capable of producing radiation by itself, a substance, which iscapable of emitting light by itself, a substance, which is capable offorming a color by itself, or a substance, which is capable of producingfluorescence by itself when being exposed to light. Alternatively, thelabeling substance may be a substance, which is capable of causing achemical substance to emit light, to form a color, or to produce thefluorescence through, for example, decomposition or reaction of thechemical substance when being brought into contact with the chemicalsubstance. As for the former type of the labeling substance, aradioactive isotope may be employed as the radiation producing labelingsubstance. Also, an acridinium ester, or the like, may be employed asthe light emitting labeling substance. Further, gold colloidalparticles, or the like, may be employed as the color forming labelingsubstance. Furthermore, fluorescein, or the like, may be employed as thefluorescent labeling substance. As the latter type of the labelingsubstance, an enzyme may be employed. Examples of the enzymes includealkaline phosphatase, peroxidase, luciferase, and β-galactosidase. Whenone of the above-enumerated enzymes acting as the labeling substance isbrought into contact with a chemical luminescence substrate, a dyesubstrate, or a fluorescence substrate, the enzyme is capable of causingthe chemical luminescence substrate to produce the chemicalluminescence, causing the dye substrate to form a color, or causing thefluorescence substrate to produce the fluorescence.

By way of example, in cases where the enzyme is alkaline phosphatase,peroxidase, or luciferase, the chemical luminescence substrate may bedioxetane, luminol, or luciferin, respectively. In cases where theenzyme is alkalinephosphatase, the dye substrate may be p-nitrophenylphosphate. In cases where the enzyme is β-galactosidase, the dyesubstrate may be p-nitrophenyl-β-D-galactoside, or the like. In caseswhere the enzyme is alkaline phosphatase, the fluorescence substrate maybe 4-methylumbellifer phosphoric acid. In cases where the enzyme isperoxidase, the fluorescence substrate may be3-(4-hydroxyphenyl)-propionic acid. In cases where the enzyme isβ-galactosidase, the fluorescence substrate may be4-methylumbellifer-β-D-galactoside, or the like.

In a second aspect, the biochemical analysis unit in accordance with thepresent invention is applicable to an assay process for:

i) obtaining a biochemical analysis unit provided with a plurality ofporous adsorptive regions, to which ligands or receptors have been boundrespectively,

ii) subjecting a reaction liquid containing at least one kind of areceptor or at least one kind of a ligand to specific binding with theligands or the receptors, each of which has been bound to one of theporous adsorptive regions of the biochemical analysis unit, the receptoror the ligand being thereby specifically bound to at least one of theligands, each of which has been bound to one of the porous adsorptiveregions of the biochemical analysis unit, or at least one of thereceptors, each of which has been bound to one of the porous adsorptiveregions of the biochemical analysis unit,

iii) subjecting a labeled body, which has been labeled with a labelingsubstance, to specific binding with the receptor or the ligand havingbeen specifically bound to at least one of the ligands, each of whichhas been bound to one of the porous adsorptive regions of thebiochemical analysis unit, or at least one of the receptors, each ofwhich has been bound to one of the porous adsorptive regions of thebiochemical analysis unit, and

iv) detecting the receptor or the ligand, which has been specificallybound to at least one of the ligands or at least one of the receptors.

The aforesaid second aspect of the assay process is the so-calledsandwich technique, wherein the receptor or the ligand, which is to bedetected, is sandwiched between the ligand or the receptor, which hasbeen bound to the adsorptive region, and the labeled body. In this case,the receptor or the ligand, which is to be detected, is the substance,which has been sampled from an organism through extraction, isolation,or the like, or has been subjected to chemical treatment after beingsampled, and which has been labeled with the labeling substance. Thereceptor or the ligand is capable of undergoing the specific bindingwith at least one of the ligands, each of which has been bound to one ofthe porous adsorptive regions of the biochemical analysis unit, or atleast one of the receptors, each of which has been bound to one of theporous adsorptive regions of the biochemical analysis unit. Examples ofthe receptors or the ligands, which are to be detected, includehormones, tumor markers, enzymes, antibodies, antigens, abzymes, otherproteins, nucleic acids, DNA's, and mRNA's.

The labeled body, which has been labeled with the labeling substance, isa body, which has been labeled with the labeling substance describedabove and is capable of undergoing the specific binding with a reactionsite of the receptor or the ligand, which is to be detected. Examples ofthe labeled bodies include antigens, antibodies, hormones, tumormarkers, enzymes, abzymes, other proteins, nucleic acids, cDNA's, DNA's,and RNA's, whose characteristics, compositions, structures, basesequences, base lengths, and the like, are known.

In a third aspect, the biochemical analysis unit in accordance with thepresent invention is applicable to an assay process for:

i) obtaining a biochemical analysis unit provided with a plurality ofporous adsorptive regions, to which ligands or receptors have been boundrespectively,

ii) subjecting a reaction liquid containing at least one kind of anauxiliary substance-bound receptor or at least one kind of an auxiliarysubstance-bound ligand, to which an auxiliary substance has been bound,to specific binding with the ligands or the receptors, each of which hasbeen bound to one of the porous adsorptive regions of the biochemicalanalysis unit, the auxiliary substance-bound receptor or the auxiliarysubstance-bound ligand being thereby specifically bound to at least oneof the ligands, each of which has been bound to one of the porousadsorptive regions of the biochemical analysis unit, or at least one ofthe receptors, each of which has been bound to one of the porousadsorptive regions of the biochemical analysis unit,

iii) subjecting an auxiliary substance-combinable labeling substance,which is capable of undergoing specific binding with the auxiliarysubstance, to specific binding with the auxiliary substance-boundreceptor or the auxiliary substance-bound ligand having beenspecifically bound to at least one of the ligands, each of which hasbeen bound to one of the porous adsorptive regions of the biochemicalanalysis unit, or at least one of the receptors, each of which has beenbound to one of the porous adsorptive regions of the biochemicalanalysis unit, and

iv) detecting the auxiliary substance-bound receptor or the auxiliarysubstance-bound ligand, which has been specifically bound to at leastone of the ligands or at least one of the receptors.

The auxiliary substance is a substance capable of undergoing the bindingwith the auxiliary substance-combinable labeling substance. Examples ofpreferable auxiliary substances include antigens, such as digoxigenin,biotin, avidin, and fluorescein, and antibodies with respect to theabove-enumerated antigens. Also, the auxiliary substance may be abiological binding partner, such as avidin with respect to biotin. Inthis case, the auxiliary substance-combinable labeling substance is asubstance, which is capable of undergoing the specific binding with theauxiliary substance and has been labeled with the labeling substancedescribed above.

How a biochemical analysis using the biochemical analysis unit inaccordance with the present invention is performed will be describedhereinbelow by taking a chemical luminescence technique as an example.

In the chemical luminescence technique using the biochemical analysisunit in accordance with the present invention, firstly, the ligands orthe receptors are bound respectively to the adsorptive regions of thebiochemical analysis unit, which is provided with the plurality of theadsorptive regions. In cases where each of the adsorptive regions of thebiochemical analysis unit is provided with the layer, which has thepores having a comparatively small mean pore diameter, and the layer,which has the pores having a comparatively large mean pore diameter, thebinding of the ligand or the receptor with each of the adsorptiveregions is performed from the side of the layer, which has the poreshaving a comparatively small mean pore diameter. Also, in cases whereeach of the adsorptive regions of the biochemical analysis unit isprovided with the layer constituted of the material having acomparatively large quantity of the functional group, which is capableof binding with the ligand or the receptor to be bound to the adsorptiveregion, and the layer constituted of the material having a comparativelysmall quantity of the functional group, which is capable of binding withthe ligand or the receptor to be bound to the adsorptive region, thebinding of the ligand or the receptor with each of the adsorptiveregions is performed from the side of the layer constituted of thematerial having a comparatively large quantity of the functional group,which is capable of binding with the ligand or the receptor to be boundto the adsorptive region. After the ligands or the receptors have beenspotted respectively onto the adsorptive regions of the biochemicalanalysis unit, the ligands or the receptors are capable of being fixedto the adsorptive regions with ultraviolet light irradiation, or thelike.

As described above, the binding of the ligand or the receptor with eachof the adsorptive regions is performed from the side of the layer, whichhas the pores having a comparatively small mean pore diameter. Also, thebinding of the ligand or the receptor with each of the adsorptiveregions is performed from the side of the layer constituted of thematerial having a comparatively large quantity of the functional group,which is capable of binding with the ligand or the receptor to be boundto the adsorptive region. As a result, the ligand or the receptor, whichis fixed to each of the adsorptive regions, converges upon the layer,which has the pores having a comparatively small mean pore diameter.Also, the ligand or the receptor, which is fixed to each of theadsorptive regions, converges upon the layer constituted of the materialhaving a comparatively large quantity of the functional group, which iscapable of binding with the ligand or the receptor to be bound to theadsorptive region.

Thereafter, a labeled receptor or a labeled ligand, which has beenlabeled with a labeling substance, is subjected to specific binding withthe ligands or the receptors, each of which has been bound to one of theadsorptive regions of the biochemical analysis unit. In order to performthe specific binding of labeled receptor or the labeled ligand with theligands or the receptors, each of which has been bound to one of theadsorptive regions of the biochemical analysis unit, a reactor, in whicha reaction liquid is capable of being forcibly caused to flow such thatthe reaction liquid flows across each of the adsorptive regions of thebiochemical analysis unit, is utilized.

FIG. 5 is a schematic sectional view showing an example of a reactor, inwhich a reaction liquid is capable of being forcibly caused to flow.With reference to FIG. 5, the reactor comprises a reaction vessel 41, aliquid circulating pipe 42 and a pump 43. The reaction vessel 41 isprovided with a biochemical analysis unit support section 44, whichsupports a biochemical analysis unit 40 and has sealing functions forpreventing liquid leakage. A reaction vessel main body 45 of thereaction vessel 41 comprises a reaction vessel upper half 46 and areaction vessel lower half 47. The reaction vessel upper half 46 isreleasably secured to the reaction vessel main body 45. When thebiochemical analysis unit 40 is to be set within the reaction vessel 41,the reaction vessel upper half 46 is dismounted from the reaction vesselmain body 45, and the biochemical analysis unit 40 is set within thereaction vessel 41. A bottom wall of the reaction vessel lower half 47is provided with a liquid inlet 48, through which a liquid is capable offlowing. Also, a top wall of the reaction vessel upper half 46 isprovided with a liquid outlet 49, through which the liquid is capable offlowing. Further, the liquid circulating pipe 42 is releasably fitted tothe liquid inlet 48 and the liquid outlet 49 of the reaction vessel 41.The reactor is constituted such that the liquid is introduced by thepump 43 into the reaction vessel main body 45 through the liquid inlet48, passed through the biochemical analysis unit 40, discharged throughthe liquid outlet 49, and circulated through the liquid circulating pipe42.

The biochemical analysis unit 40 provided with the adsorptive regions,to which the ligands or the receptors have been bound respectively, isset in the reactor. Also, the reaction liquid containing the labeledreceptor or the labeled ligand is introduced into the reaction vessel41. Thereafter, the pump 43 is actuated, and the reaction liquid isforcibly caused to flow such that the reaction liquid flows across eachof the adsorptive regions of the biochemical analysis unit 40. In thismanner, the labeled receptor or the labeled ligand is capable of beingsubjected to the specific binding with the ligands or the receptors,which have been bound respectively to the adsorptive regions of thebiochemical analysis unit 40. As described above, the ligand or thereceptor, which is fixed to each of the adsorptive regions of thebiochemical analysis unit 40, converges upon one of the layersconstituting each of the adsorptive regions of the biochemical analysisunit 40. The biochemical analysis unit 40 may be set in the reactor suchthat the layer, upon which the fixed ligand or the fixed receptorconverges, stands facing the upstream side of the flow of the reactionliquid, which is forcibly caused to flow. Alternatively, the biochemicalanalysis unit 40 may be set in the reactor such that the layer otherthan the layer, upon which the fixed ligand or the fixed receptorconverges, stands facing the upstream side of the flow of the reactionliquid, which is forcibly caused to flow.

The reactor illustrated in FIG. 5 is constituted such that the reactionliquid is forcibly caused to flow in one direction by the pump 43.Alternatively, for example, a reactor may be constituted such that asyringe and a piston are utilized in lieu of the pump 43, and thereaction liquid is caused to undergo reciprocal flowing within thereaction vessel. As another alternative, a reactor may be utilized, inwhich the reaction liquid merely passes through the biochemical analysisunit 40 from below (or from above) and is not circulated.

In the aforesaid example, the specific binding is performed by use ofthe reactor, which is capable of forcibly causing the reaction liquid toflow such that the reaction liquid flows across each of the adsorptiveregions of the biochemical analysis unit. However, the biochemicalanalysis unit in accordance with the present invention is not limited tothe use within the reactor described above. For example, the biochemicalanalysis unit in accordance with the present invention may be utilizedfor the shaking technique, wherein the biochemical analysis unit and thereaction liquid are put into a hybridization bag, vibrations are givento the hybridization bag, and the labeled receptor or the labeled ligandis thus moved through convection or diffusion and is specifically boundto one of the ligands or the receptors having been fixed to theadsorptive regions of the biochemical analysis unit.

However, in cases where each of the adsorptive regions of thebiochemical analysis unit is provided with the layer, which has thepores having a comparatively small mean pore diameter, and the layer,which has the pores having a comparatively large mean pore diameter, thebiochemical analysis unit should preferably be set within the aforesaidreactor, which is capable of forcibly causing the reaction liquid toflow such that the reaction liquid flows across each of the adsorptiveregions of the biochemical analysis unit. In such cases, the pressuredrag of the reaction liquid within the layer, which has the pores havinga comparatively large mean pore diameter, is capable of being keptsmall, and the flow rate of the reaction liquid is capable of beingenhanced. Therefore, an enhanced reaction efficiency is capable of beingexpected.

The ligand or the receptor, which has been bound to each of theadsorptive regions of the biochemical analysis unit, converges upon oneof the layers constituting each of the adsorptive regions of thebiochemical analysis unit. Therefore, the labeled receptor or thelabeled ligand, which is bound to at least one of the ligands or thereceptors having been bound respectively to the adsorptive regions ofthe biochemical analysis unit, converges upon one of the layersconstituting each of the adsorptive regions of the biochemical analysisunit.

In order for the labeled receptor or the labeled ligand, which has notbeen specifically bound to the ligands or the receptors having beenbound respectively to the porous adsorptive regions of the biochemicalanalysis unit, to be removed, the biochemical analysis unit having beenset within the reaction vessel should preferably be washed with atechnique for forcibly causing a washing liquid to flow across each ofthe adsorptive regions. In such cases, since the washing liquid isforcibly caused to flow across each of the adsorptive regions, thelabeled receptor or the labeled ligand, which has not been specificallybound to the ligands or the receptors having been bound respectively tothe porous adsorptive regions of the biochemical analysis unit, iscapable of being peeled off and removed efficiently. Therefore, thewashing efficiency is capable of being enhanced markedly.

After a reaction liquid, which contains an enzyme-labeled antibodydescribed later, is forcibly caused to flow such that the reactionliquid flows across each of the adsorptive regions of the biochemicalanalysis unit, and the enzyme-labeled antibody is thus subjected to thespecific binding with the labeled receptor or the labeled ligand, theenzyme-labeled antibody, which has not been specifically bound to thelabeled receptor or the labeled ligand, may be removed. In cases wherethe enzyme-labeled antibody, which has not been specifically bound tothe labeled receptor or the labeled ligand, is to be removed, thewashing process described above should preferably be performed. In thismanner, the enzyme-labeled antibody, which has not been specificallybound to the labeled receptor or the labeled ligand, is capable of beingpeeled off and removed efficiently. Therefore, the washing efficiency iscapable of being enhanced markedly.

Before the enzyme-labeled antibody is subjected to the specific bindingwith the labeled receptor or the labeled ligand having been specificallybound to at least one of the ligands, each of which has been bound toone of the adsorptive regions of the biochemical analysis unit, or atleast one of the receptors, each of which has been bound to one of theadsorptive regions of the biochemical analysis unit, the adsorptiveregions should preferably be blocked with a blocking process, wherein ablocking buffer with respect to the enzyme-labeled antibody is forciblycaused to flow such that the blocking buffer flows across each of theadsorptive regions. With the blocking process, the problems are capableof being prevented from occurring in that, instead of the enzyme-labeledantibody being subjected to the specific binding with the antigen of thelabeled receptor or the labeled ligand, the enzyme-labeled antibody isdirectly bound to the adsorptive regions of the biochemical analysisunit.

Thereafter, the reaction liquid, which contains the enzyme-labeledantibody, is forcibly caused to flow such that the reaction liquid flowsacross each of the adsorptive regions of the biochemical analysis unit,and the enzyme-labeled antibody is thus subjected to the specificbinding with the labeled receptor or the labeled ligand. Theenzyme-labeled antibody is the antibody with respect to the labelingsubstance of the labeled receptor or the labeled ligand, which antibodyhas been labeled with an enzyme. (In cases where the labeling substanceof the labeled receptor or the labeled ligand is an antibody, theenzyme-labeled antibody is the antigen with respect to the labelingsubstance of the labeled receptor or the labeled ligand, which antigenhas been labeled with an enzyme.)

After the enzyme-labeled antibody has thus been subjected to thespecific binding with the labeled receptor or the labeled ligand, thewashing liquid is forcibly caused to flow across each of the adsorptiveregions of the biochemical analysis unit, and the enzyme-labeledantibody, which has not been specifically bound to the labeled receptoror the labeled ligand, is thereby removed. Thereafter, a chemicalluminescence substrate is fed into the adsorptive regions and thusbrought into contact with the enzyme-labeled antibody, which has beenspecifically bound to the labeled receptor or the labeled ligand.

In cases where the chemical luminescence substrate and the enzyme arebrought into contact with each other, the chemical luminescence havingwavelengths falling within the visible light wavelength range isproduced. Therefore, the produced chemical luminescence may be detectedphotoelectrically from the side of the biochemical analysis unit, onwhich side the ligands or the receptors having been bound to theadsorptive regions have converged. The image data for a biochemicalanalysis may thus be formed in accordance with the detected chemicalluminescence. In this manner, the labeled receptor or the labeled ligandis capable of being detected and determined without the signal comingfrom the labeling substance being attenuated.

The present invention will further be illustrated by the followingnonlimitative examples.

EXAMPLES Example 1

With an etching technique, 2,500 fine holes were formed in a SUS304sheet (acting as a base plate material sheet) having a size of 50 mm×50mm and a thickness of 100 μm. Each of the fine holes had a circularopening region having a hole diameter of 0.3 mm. The fine holes wereformed at a hole pitch of 0.45 mm.

Thereafter, as filling materials to be filled in adsorptive regions, twokinds of 6,6-nylons having different polymerization degrees (hereinafterreferred to as the high-polymerization-degree 6,6-nylon and thelow-polymerization-degree 6,6-nylon), which were supplied by AldrichCo., were prepared. A solution of the high-polymerization-degree6,6-nylon was prepared by dissolving 10 g of thehigh-polymerization-degree 6,6-nylon in 52 g of formic acid. Also, asolution of the low-polymerization-degree 6,6-nylon was prepared bydissolving 10 g of the low-polymerization-degree 6,6-nylon in 52 g offormic acid. The thus prepared solution of thehigh-polymerization-degree 6,6-nylon had a viscosity of 6.25 Pa·s, andthe solution of the low-polymerization-degree 6,6-nylon had a viscosityof 0.94 Pa·s. Thereafter, 15 g of deionized water was added per 85 g ofthe solution of the high-polymerization-degree 6,6-nylon. Also, 15 g ofdeionized water was added per 85 g of the solution of thelow-polymerization-degree 6,6-nylon. Thereafter, the solution of thelow-polymerization-degree 6,6-nylon was cast to uniform wet thicknessonto a clean glass plate. The solution of the high-polymerization-degree6,6-nylon was then cast to uniform wet thickness onto the cast layer ofthe solution of the low-polymerization-degree 6,6-nylon. The wetthickness of the cast layer of the solution of thelow-polymerization-degree 6,6-nylon was 300 μm. The wet thickness of thecast layer of the solution of the high-polymerization-degree 6,6-nylonwas 100 μm. The total wet thickness of the combination of the cast layerof the solution of the low-polymerization-degree 6,6-nylon and the castlayer of the solution of the high-polymerization-degree 6,6-nylon wasthus 400 μm.

Thereafter, the combination of the cast layer of the solution of thelow-polymerization-degree 6,6-nylon and the cast layer of the solutionof the high-polymerization-degree 6,6-nylon was dipped in an aqueousformic acid solution containing formic acid and deionized water in aformic acid:deionized water ratio of 45:55. In this manner, fine poreswere formed in the cast layer of the solution of thelow-polymerization-degree 6,6-nylon and the cast layer of the solutionof the high-polymerization-degree 6,6-nylon. The combination of the castlayer of the solution of the low-polymerization-degree 6,6-nylon and thecast layer of the solution of the high-polymerization-degree 6,6-nylon,in which the fine pores had been formed, were then dried. Observation ofa film cross-section of the combination of the two cast layers revealedthat a layer having comparatively large pores (with diameters ofapproximately 1 μm) was formed from the cast layer of the solution ofthe low-polymerization-degree 6,6-nylon, and a layer havingcomparatively small pores (with diameters of approximately 0.4 μm) wasformed from the cast layer of the solution of thehigh-polymerization-degree 6,6-nylon. (The layer having comparativelysmall pores, which layer had been formed from the cast layer of thesolution of the high-polymerization-degree 6,6-nylon, will hereinbelowbe referred to as the finer pore layer.) The thickness of the thusobtained film having the multi-layer structure was 180 μm.

Thereafter, an adhesive agent was applied to one surface of the baseplate described above, and the adhesive agent, which entered into theholes having been formed in the base plate, was removed by suction. Theadhesive agent remaining on the surface of the base plate was thendried. Thereafter, the adsorptive region forming material having themulti-layer structure described above was pressed against the surface ofthe base plate, which surface had been coated with the adhesive agent.The adsorptive region forming material having the multi-layer structuredescribed above was thus press-fitted into the fine holes of the baseplate and laminated with the surface of the base plate, which surfacehad been coated with the adhesive agent. The press-fitting process wasperformed with a calendering technique at a pressure of 100 kPa/cm. (Thetemperature of one of calendering roll was set at 150° C., and thetemperature of the other calendering roll was set at 50° C.) In thismanner, a biochemical analysis unit was prepared.

Also, 10 nl of a pBR328-DNA liquid having a concentration of 25 ng/μl(supplied by Roche Diagnostics K.K.), in which the pBR328-DNA had beenconverted into a single stranded form by thermal denaturation, wasspotted onto each of the adsorptive regions of the biochemical analysisunit having been prepared in the manner described above. The spottingwas performed from the side of the finer pore layer of each of theadsorptive regions of the biochemical analysis unit. Thereafter, withirradiation of ultraviolet light (254 nm, 33 mJ/cm²), the singlestranded pBR328-DNA was fixed to the adsorptive regions of thebiochemical analysis unit.

Thereafter, 71 g of disodium hydrogen phosphate (anhydrous) wasdissolved in 800 ml of deionized water having been sterilized, and thepH value of the resulting solution was adjusted at 7.2 by the additionof 3 ml to 4 ml of phosphoric acid. Also, the total volume was made upto 1,000 ml by the addition of deionized water having been sterilized.In this manner, a 1M phosphoric acid buffer was prepared. Thereafter, 7g of a dodecyl sulfonic acid sodium salt was added to 50 ml of the thusprepared phosphoric acid buffer and 43 ml of deionized water having beensterilized. The resulting mixture was heated, and the dodecyl sulfonicacid sodium salt was dissolved with stirring. Also, 200 μl of a 0.5MEDTA was added. In this manner, a hybridization solution was prepared.

Also, a pBR328-DNA liquid (supplied by Roche Diagnostics K.K.), whichhad been labeled with digoxigenin (DIG) and had a concentration of 5ng/μl, was diluted with a TE buffer solution (a mixed solution of 10 mMof Tris-HCL and 1 mM of EDTA, supplied by Nippon Gene K.K.). TheDIG-labeled pBR328-DNA liquid was then subjected to thermaldenaturation, and the DIG-labeled pBR328-DNA was thus converted into asingle stranded form. Thereafter, the DIG-labeled pBR328-DNA liquid wasdiluted with the hybridization solution described above. In this manner,a hybridization reaction liquid, which contained the DIG-labeledpBR328-DNA at a concentration of 10 pg/ml, was prepared.

Thereafter, the biochemical analysis unit described above was put in ahybridization bag, and 10 ml of the hybridization reaction liquid wasintroduced into the hybridization bag. The biochemical analysis unit wasthus subjected to a hybridization reaction at a temperature of 68° C.for 18 hours with a shaking technique. After the hybridization reactionwas performed, a washing liquid was fed into the hybridization bag, andthe biochemical analysis unit was washed with the washing liquid.

Also, a washing buffer (supplied by Roche Diagnostics K.K.) was dilutedwith sterilized deionized water to a concentration of 1/10, and awashing liquid for chemical luminescence was thereby prepared. The thusprepared washing liquid for chemical luminescence was introduced intothe hybridization bag, in which the biochemical analysis unit had beenaccommodated, and a shaking operation was performed for five minutes.

Thereafter, by use of a maleic acid buffer (supplied by RocheDiagnostics K.K.), which had been diluted with sterilized deionizedwater to a concentration of 1/10, a blocking buffer solution (suppliedby Roche Diagnostics K.K.) was diluted to a concentration of 1/10. Thethus diluted blocking buffer solution was then subjected to filtrationwith a polyether sulfone filter (pore diameter: 0.2 μm) and thenutilized as a blocking agent. The blocking agent was fed into thehybridization bag, from which the washing liquid had been discharged. Ashaking operation was then performed for one hour, and a blockingreaction was thus performed.

Thereafter, an anti-digoxigenin-AP-conjugate (an alkalinephosphatase-labeled digoxigenin antibody, supplied by Roche DiagnosticsK.K.) was subjected to centrifugal filtration with a polyvinylidenefluoride filter (pore diameter: 0.2 μm). Theanti-digoxigenin-AP-conjugate having been collected by filtration wasthen diluted with the aforesaid blocking agent to a concentration of0.75 U/ml, and an enzyme-labeled antibody liquid was thereby prepared.Thereafter, 5 ml of the thus prepared enzyme-labeled antibody liquid wasfed into the hybridization bag, from which the blocking agent had beendischarged. A shaking operation was then performed for one hour, and anantigen-antibody reaction was thus performed.

After the antigen-antibody reaction was completed, the aforesaid washingliquid for chemical luminescence was fed into the hybridization bag, andthe biochemical analysis unit was washed with the washing liquid forchemical luminescence. The biochemical analysis unit was taken out fromthe hybridization bag and was then brought into contact with a liquidcontaining a chemical luminescence substrate (CDP-star, ready to use,supplied by Roche Diagnostics K.K.). Also, the chemical luminescence,which was emitted from the adsorptive regions of the biochemicalanalysis unit, was detected photoelectrically by use of a cooled CCDcamera (LAS1000, supplied by Fuji Photo Film Co., Ltd.). Thephotoelectric detection of the chemical luminescence was performed fromthe side of the finer pore layer of each of the adsorptive regions ofthe biochemical analysis unit. In this manner, a digital signal wasformed.

Example 2

The chemical luminescence operations were performed in the same manneras that in Example 1, except that adsorptive regions of a biochemicalanalysis unit were formed such that the wet thickness of the cast layerof the solution of the low-polymerization-degree 6,6-nylon was 200 μm,the wet thickness of the cast layer of the solution of thehigh-polymerization-degree 6,6-nylon was 200 μm, and the total wetthickness of the combination of the cast layer of the solution of thelow-polymerization-degree 6,6-nylon and the cast layer of the solutionof the high-polymerization-degree 6,6-nylon was thus 400 μm. Also, adigital signal was formed in the same manner as that in Example 1.

Example 3

The chemical luminescence operations were performed in the same manneras that in Example 1, except that adsorptive regions of a biochemicalanalysis unit were formed such that the wet thickness of the cast layerof the solution of the low-polymerization-degree 6,6-nylon was 100 μm,the wet thickness of the cast layer of the solution of thehigh-polymerization-degree 6,6-nylon was 300 μm, and the total wetthickness of the combination of the cast layer of the solution of thelow-polymerization-degree 6,6-nylon and the cast layer of the solutionof the high-polymerization-degree 6,6-nylon was thus 400 μm. Also, adigital signal was formed in the same manner as that in Example 1.

Example 4

A pBR328-DNA liquid, which had been labeled with DIG and had aconcentration of 5 ng/μl, was diluted with a TE buffer solution. TheDIG-labeled pBR328-DNA liquid was then subjected to thermaldenaturation, and the DIG-labeled pBR328-DNA was thus converted into asingle stranded form. Thereafter, the DIG-labeled pBR328-DNA liquid wasdiluted with a hybridization solution, which was prepared in the samemanner as that in Example 1. In this manner, a hybridization reactionliquid, which contained the DIG-labeled pBR328-DNA at a concentration of1 pg/ml, was prepared.

Thereafter, a biochemical analysis unit, which had been prepared in thesame manner as that in Example 2, and in which the single strandedpBR328-DNA had been fixed to the adsorptive regions, was set in thereaction vessel of the reactor illustrated in FIG. 5. Also, 4 ml of thehybridization reaction liquid was fed into the reaction vessel, in whichthe biochemical analysis unit had been accommodated. The pump of thereactor was actuated, and a hybridization reaction was performed at atemperature of 68° C. for 18 hours. After the hybridization reaction wasfinished, the pump was actuated, and the adsorptive regions of thebiochemical analysis unit were washed.

Thereafter, the washing liquid for chemical luminescence, which wasprepared in Example 1, was fed into the reaction vessel, in which thebiochemical analysis unit had been accommodated. The pump was thenactuated for five minutes, and the liquid in the adsorptive regions ofthe biochemical analysis unit was thus replaced by the washing liquidfor chemical luminescence. Thereafter, the washing liquid was dischargedfrom the reaction vessel, and the blocking agent, which had beenprepared in Example 1, was fed into the reaction vessel. The pump wasthen driven for 10 minutes. In this manner, the liquid at all parts ofthe adsorptive regions of the biochemical analysis unit was replaced bythe blocking agent. Thereafter, the operation of the pump was ceased,and the blocking agent was allowed to stand for 50 minutes within thereaction vessel.

Thereafter, the blocking agent was discharged from the reaction vessel,and 5 ml of the enzyme-labeled antibody liquid, which was prepared inExample 1, was fed into the reaction vessel. The pump was then drivenfor one minute. In this manner, the liquid at all parts of theadsorptive regions of the biochemical analysis unit was replaced by theenzyme-labeled antibody liquid. Thereafter, the operation of the pumpwas ceased, and the enzyme-labeled antibody liquid was allowed to standfor one hour within the reaction vessel.

After the antigen-antibody reaction was completed, the washing bufferdescribed above was fed into the reaction vessel. Also, the pump wasdriven, and the biochemical analysis unit was thus washed. Thereafter,the pump was driven, and the chemical luminescence substrate (CDP-star,ready to use, supplied by Roche Diagnostics K.K.) was brought intocontact with the adsorptive regions of the biochemical analysis unit.Also, the chemical luminescence, which was emitted from the adsorptiveregions of the biochemical analysis unit, was detected photoelectricallyby use of the cooled CCD camera (LAS1000, supplied by Fuji Photo FilmCo., Ltd.). The photoelectric detection of the chemical luminescence wasperformed from the side of the finer pore layer of each of theadsorptive regions of the biochemical analysis unit. In this manner, adigital signal was formed.

Comparative Example 1

A solution of the high-polymerization-degree 6,6-nylon, which wasemployed in Example 1, was prepared in the same manner as that inExample 1. A biochemical analysis unit was then prepared in the samemanner as that in Example 1, except that the solution of thehigh-polymerization-degree 6,6-nylon was uniformly cast onto a cleanglass plate, such that the wet thickness of the cast layer of thesolution of the high-polymerization-degree 6,6-nylon was 400 μm, and theadsorptive regions were thereby constituted. By use of the thus preparedbiochemical analysis unit, the chemical luminescence operations wereperformed in the same manner as that in Example 1. Also, a digitalsignal was formed in the same manner as that in Example 1.

Comparative Example 2

A solution of the low-polymerization-degree 6,6-nylon, which wasemployed in Example 1, was prepared in the same manner as that inExample 1. A biochemical analysis unit was then prepared in the samemanner as that in Example 1, except that the solution of thelow-polymerization-degree 6,6-nylon was uniformly cast onto a cleanglass plate, such that the wet thickness of the cast layer of thesolution of the low-polymerization-degree 6,6-nylon was 400 μm, and theadsorptive regions were thereby constituted. By use of the thus preparedbiochemical analysis unit, the chemical luminescence operations wereperformed in the same manner as that in Example 1. Also, a digitalsignal was formed in the same manner as that in Example 1.

Comparative Example 3

The chemical luminescence operations were performed by use of thereactor illustrated in FIG. 5 and in the same manner as that in Example4, except that a biochemical analysis unit prepared in the same manneras that in Comparative Example 1 was employed. Also, a digital signalwas formed in the same manner as that in Example 4.

With each of the biochemical analysis units formed in Examples 1, 2, 3and Comparative Examples 1 and 2, the intensity of the signal, theintensity of a background, and the signal-to-noise ratio (S/N ratio)listed in Table 1 below were obtained. TABLE 1 Well thickness Wellthickness of low- of high- polymerization- polymerization- degree degree6,6-nylon 6,6-nylon Back- S/N (μm) (μm) Signal ground ratio Example 1300 100 547300 8600 63.6 Example 2 200 200 466800 6600 70.7 Example 3100 300 437300 6900 63.4 Comp. Ex. 1 None 400 378400 11500 32.9 Comp.Ex. 2 400 None 351100 23000 15.3

As clear from Table 1, with each of the biochemical analysis unitsformed in Examples 1, 2, and 3 in accordance with the present invention,wherein each of the adsorptive regions of the biochemical analysis unitis constituted of the layer, which has the pores having a comparativelysmall mean pore diameter, and the layer, which has the pores having acomparatively large mean pore diameter, the intensity of the digitalsignal is higher than the intensity of the digital signal obtained witheach of the biochemical analysis units formed in Comparative Examples 1and 2, wherein each of the adsorptive regions of the biochemicalanalysis unit is constituted of the layer, which has the pores having asingle same mean pore diameter, and the intensity of the background waslower than the intensity of the background obtained with each of thebiochemical analysis units formed in Comparative Examples 1 and 2. Witheach of the biochemical analysis units formed in Examples 1, 2, and 3 inaccordance with the present invention, the pBR328-DNA (acting as theligand or the receptor), which has been fixed to each of the adsorptiveregions of the biochemical analysis unit, converges upon the finer porelayer (i.e., the layer, which has the pores having a comparatively smallmean pore diameter). Also, the DIG-labeled pBR328-DNA (acting as thereceptor or the ligand), which has been bound specifically to thepBR328-DNA having been fixed to each of the adsorptive regions of thebiochemical analysis unit, converges upon the finer pore layer.Therefore, the chemical luminescence, which is produced when thealkaline phosphatase-labeled digoxigenin antibody, which has been boundto the DIG-labeled pBR328-DNA, is brought into contact with the chemicalluminescence substrate (CDP-star), converges upon the finer pore layer.The thus produced chemical luminescence is detected from the side of thefiner pore layer of each of the adsorptive regions of the biochemicalanalysis unit. Therefore, the receptor or the ligand is capable of beingdetected without the signal being attenuated.

Also, with each of the biochemical analysis units formed in Examples 1,2, and 3 in accordance with the present invention, at the underside arealocated under the base plate, the layers, which constitute each of theadsorptive regions are connected with the layers, which constitute anadjacent adsorptive region. Therefore, there is possibility of thesignal propagating from a certain hole toward the adjacent hole.However, with each of the biochemical analysis units formed in Examples1, 2, and 3 in accordance with the present invention, the pBR328-DNA,which has been fixed to each of the adsorptive regions, converges uponthe finer pore layer. Therefore, the amount of the signal propagatingfrom a certain hole toward the adjacent hole is capable of being keptsmaller than with each of the biochemical analysis units formed inComparative Examples 1 and 2, wherein the pBR328-DNA is bound in adispersed form over the entire area of the adsorptive region.Accordingly, with each of the biochemical analysis units formed inExamples 1, 2, and 3 in accordance with the present invention, theintensity of the background is capable of being kept low.

With each of the biochemical analysis units formed in Example 4 andComparative Example 3, the intensity of the signal, the intensity of thebackground, and the signal-to-noise ratio (S/N ratio) listed in Table 2below were obtained. TABLE 2 Well thickness Well thickness of low- ofhigh- polymerization- polymerization- degree degree 6,6-nylon 6,6-nylonBack- S/N (μm) (μm) Signal ground ratio Example 4 200 200 259120 6320 41Comp. Ex. 3 None 400 263500 10540 25

In each of Example 4 and Comparative Example 3, the chemicalluminescence operations are performed by use of the reactor illustratedin FIG. 5, wherein the reaction liquid is forcibly caused to flow suchthat the reaction liquid flows across each of the adsorptive regions ofthe biochemical analysis unit. In such cases, as shown in Table 2, withthe biochemical analysis unit formed in Example 4 in accordance with thepresent invention, wherein each of the adsorptive regions of thebiochemical analysis unit is constituted of the layer, which has thepores having a comparatively small mean pore diameter, and the layer,which has the pores having a comparatively large mean pore diameter, theintensity of the digital signal is higher than the intensity of thedigital signal obtained with the biochemical analysis unit formed inComparative Example 3, wherein each of the adsorptive regions of thebiochemical analysis unit is constituted of the layer, which has thepores having a single same mean pore diameter, and the intensity of thebackground was lower than the intensity of the background obtained withthe biochemical analysis unit formed in Comparative Example 3.

In each of the biochemical analysis units formed in Examples 1, 2, 3,and 4 in accordance with the present invention, each of the adsorptiveregions of the biochemical analysis unit is constituted of the layer,which has the pores having a comparatively small mean pore diameter, andthe layer, which has the pores having a comparatively large mean porediameter. The same effects as those obtained in Examples 1, 2, 3, and 4in accordance with the present invention are also capable of beingobtained in cases where a biochemical analysis unit is employed, whereineach of the adsorptive regions is provided with a layer constituted of amaterial having a comparatively large quantity of a functional group,which is capable of binding with a ligand or a receptor to be bound tothe adsorptive region, and a layer constituted of a material having acomparatively small quantity of a functional group, which is capable ofbinding with the ligand or the receptor to be bound to the adsorptiveregion.

1. A biochemical analysis unit, comprising: i) a base plate, which has aplurality of holes, and ii) a porous adsorptive material, which isfilled in each of the plurality of the holes of the base plate and formseach of a plurality of adsorptive regions, wherein each of theadsorptive regions is provided with a layer constituted of a materialhaving a comparatively large quantity of a functional group, which iscapable of binding with a ligand or a receptor to be bound to theadsorptive region, and a layer constituted of a material having acomparatively small quantity of a functional group, which is capable ofbinding with the ligand or the receptor to be bound to the adsorptiveregion.
 2. A biochemical analysis unit as defined in claim 1 wherein thelayers, which constitute each of the adsorptive regions, are connectedwith the layers, which constitute an adjacent adsorptive region, at oneof surfaces of the base plate, and the biochemical analysis unit furthercomprises a signal absorbing layer for absorbing a signal, which passesthrough layers located under the base plate and thus propagates from acertain hole of the base plate toward an adjacent hole of the baseplate.
 3. A biochemical analysis unit as defined in claim 2 wherein, incases where a density of the functional group in the layer constitutedof the material having a comparatively large quantity of the functionalgroup is taken as 1, the density of the functional group in the layerconstituted of the material having a comparatively small quantity of thefunctional group is at most 0.7.
 4. A biochemical analysis unit asdefined in claim 1 wherein, in cases where a density of the functionalgroup in the layer constituted of the material having a comparativelylarge quantity of the functional group is taken as 1, the density of thefunctional group in the layer constituted of the material having acomparatively small quantity of the functional group is at most 0.7. 5.A biochemical analysis unit as defined in claim 1 wherein the base plateis constituted of a material having radiation attenuating propertiesand/or light attenuating properties.
 6. A biochemical analysis unit asdefined in claim 2 wherein the base plate is constituted of a materialhaving radiation attenuating properties and/or light attenuatingproperties.
 7. A biochemical analysis unit as defined in claim 3 whereinthe base plate is constituted of a material having radiation attenuatingproperties and/or light attenuating properties.
 8. A biochemicalanalysis unit as defined in claim 4 wherein the base plate isconstituted of a material having radiation attenuating properties and/orlight attenuating properties.